Rheumatoid arthritis (RA) is a debilitating disease that causes inflammation and deforming joint destruction. RA afflicts 1.5 million people in the United States and is becoming increasingly more prevalent in the veteran community as this population ages. It is also one of the most expensive illnesses to treat, resulting in hundreds of millions of dollars in increased health care spending by the Veteran Administration (VA). T cell dysfunction plays a key role in driving RA pathogenesis. Past studies have identified premature aging of CD4+ T cells in RA patients. Despite being called senescent, these cells are highly pro-inflammatory and produce high levels of TNF?, IFN?, and cytolytic granules. There are still many unanswered questions about senescent CD4+ T cells. In particular, the mechanisms that accelerate T cell senescence and the effector functions that contribute to autoimmunity remain poorly defined. Understanding the etiology and the impact of premature T cell senescence in RA may lead to new approaches to monitor and treat this currently incurable chronic disease. In recently published data, we identified an abundance of memory CD4+ T cells specific for viral antigens in adults who have never been infected with these viruses. Intriguingly, these T cells can be activated in vitro by similar peptides from common microbes, indicating that a subset of CD4+ memory cells is generated predominately by cross-reactive microbial and/or environmental antigen stimulation. In new preliminary studies, we found that many memory T cells generated by cross-reactive stimulation are Ki67 positive, suggesting that these cells receive continuous stimulatory signals. Because continuous or repetitive TCR stimulation has the potential to drive senescence, we hypothesize that chronic stimulation by common environmental antigens contributes to the development of senescent CD4+ T cells. In new preliminary studies, we have begun to investigate the biology of senescence in RA patients. We identified several distinct CD4+ T cell subsets, including a CD5 high population that produces the greatest level of perforin. High CD5 expression suggests a stronger avidity for self-antigens. We thus hypothesize that senescent CD4+ T cells are heterogeneous and contain subsets that are self-reactive and their cytolytic activity contributes to RA pathogenesis. The proposed study has two interrelated yet independent objectives. The first is to define the functional heterogeneity of senescent CD4+ T cells in RA patients and test the hypothesis that the most cytolytic senescent subsets are preferentially expanded in patients with active disease. We will investigate the full diversity of senescent T cells in RA patients using cytometry by time-of-flight (CyTOF). CyTOF measures the expression of more than 40 proteins at a single cell level, and thus it is an ideal tool for discovering new cellular function. The second objective is to determine whether senescent T cells from RA patients are more cross-reactive than nonsenescent T cells from the same individual and if these cells have the ability to recognize self-antigens relevant in RA. The breadth of cross-reactivity will be determined in a systematic approach using a yeast library that displays over 108 random peptide-MHC complexes. Whether senescent T cells can recognize self-peptides from putative RA autoantigens will be determined by direct binding to peptide- MHC complexes using tetramers and by assaying functional response to antigen stimulation. Data generated from this study will provide the fundamental information necessary to develop new disease biomarkers and for designing tolerizing approaches to combat premature senescence in RA patients.